Title: Mapping the Interactions of Alpha-Synuclein to Lipid Membranes in the Physiological Limit

Abstract: The discovery that alpha-synuclein, an intrinsically disordered protein, was linked to a multitude of neurodegenerative diseases (‘‘synucleinopathies’’) prompted intense efforts to understand the function of alpha-synuclein. While many hypotheses centrally involve the ability of alpha-synuclein to bind to highly-curved lipid membranes, it is difficult to reconcile the diversity of sub-neuronal organelles (and corresponding lipid membranes) with which alpha-synuclein is purported to interact. To that end, we have pioneered a generalizable technique to encapsulate uniform spherical-nanoparticles with a single membrane (with lipid composition of our choosing), creating an in vitro probe with precise membrane curvature in a previously un-realizable biological limit ( 35-50 nm). Using isothermal titration calorimetry we report the binding affinities of alpha-synuclein variants as a function of increasing membrane curvature. While previous measurements required an un-physiological high mol% of anionic lipids to recapitulate strong alpha-synuclein binding, we demonstrate the complex interplay between lipid charge, lipid intrinsic curvature, and membrane curvature to tease out the biochemical and biophysical parameters that drive alpha-synuclein binding. These parameters will be correlated to states in the dynamic lipid composition of sub-neuronal organelles, providing the data necessary to develop hypotheses on the molecular action and dysfunction of alpha-synuclein within the neuron.